Turkish Journal of Entomology

98 Pages·2015·4.05 MB·Turkish

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Türkiye Entomoloji Dergisi
(Turkish Journal of Entomology)

Cilt (Vol.) 39 Sayı (No.) 2 Haziran (June) 2015

İnceleme ve Değerlendirmede Bilimsel Olarak Katkıda Bulunanlar
(Scientific Advisory Board)

ADAMSKI, Z., Poland KEPENEKÇİ, İlker, Ankara
AKBULUT, Mikail, Kayseri KESKİN, Bekir, İzmir
AKYAZI, Faruk, Ordu KHANJANI, M., Iran
AMBROZIAK, A. K., Poland KIVAN, Müjgan, Tekirdağ
ARMENGAUD, C., France KOÇAK, Erhan, Isparta
ASSING, V., Germany KORNEYEV, V. Ukraine
ATLIHAN, Remzi, Van KUBIC, S., Czech Republic)
AYDIN, Gökhan, Isparta KUMRAL, Nabi Alper, Bursa
AZİZOĞLU, Uğur, Erciyes LEGALOV, A. A., Russia
BAŞHAN, Mehmet, Diyarbakır LIETTI, M. M., Argentina
BAYRAM, Ahmet, Diyarbakır MÁCA, J., Czech Republic
BORDERA, S., Spain MENNAN, Sevilhan, Samsun
BUTT, T., UK MERTLIK, J., USA
BÜYÜKGÜZEL, Ender, Zonguldak NAYAK, M. K., Australia
BÜYÜKGÜZEL, Kemal, Zonguldak OOSTERBROEK, P., Holland
CANHİLAL, Ramazan, Erciyes OPERMAN, C. H., USA
CİVELEK, Hasan Sungur, Muğla ÖZBEK, Hikmet, Erzurum
ÇAKMAK, İbrahim, Aydın ÖZDER, Nihal, Tekirdağ
ÇALMAŞUR, Önder, Erzurum ÖZGEN, İnanç, Elazığ
ÇETİNTAŞ, Ramazan, Kahramanmaraş ÖZGÖKÇE, Salih, Van
ÇIKMAN, Emine, Şanlıurfa ÖZSEMERCİ, Fatma, İzmir
DAĞLI, Fatih, Antalya PELLIZZARİ, G., Italy
DEVRAN, Zübeyir, Antalya PLATIA, G., Italy
DOĞAN, Salih, Erzincan SAĞLAM, Özgür, Tekirdağ
DURMUŞOĞLU, Enver, İzmir SANCHEZ-PENA, S., Mexico
DURSUN, Ahmet, Amasya SARIKAYA, Oğuzhan, Isparta
ELMA Fatmanur, Konya SERTKAYA, Erdal, Hatay
ELMALI, Meryem, Konya SHAPIRO-ILAN, D., USA
ER, Mehmet Kubilay, Kahramanmaraş SOLODOVNIKOV, A., Denmark
ERDEM, Meltem, Zonguldak SÖĞÜT, Mehmet Ali, Isparta
ERLER, Fedai, Antalya STANLEY D. W., USA
EROĞLU, Mahmut, Trabzon SULLIVAN, Sebahat, Samsun
FERİZLİ, Ahmet Güray, Ankara TEZCAN, Serdar, İzmir
FRIEDBERG, A., Israel TOKTAY, Halil, Niğde
GARONNA, A. P., Italy TOMONOVIC, Z., Serbia
GAUGLER, R. USA TOZLU, Göksel, Erzurum
GÖKÇE, Ayhan, Niğde TUNCA, Hilal, Ankara
GÖZEL, Uğur, Çanakkale TUNÇBİLEK, Aydın Şüzü, Kayseri
GÜRBÜZ, Mehmet Faruk, Isparta ÜNLÜ, Levent, Konya
HAMBÄCK, P., Sweden
VAVRE, F., France
HYRŠL, P., Czech Republic
VELİOĞLU, Sibel, Ankara
IŞIKBER, Ali Arda, Kahramanmaraş
WANI, A. A., India
JAPOSHVILI, George, Isparta
YANAR Dürdane, Tokat
JUSSILA, R., Finland
YEFREMOVA, Z., Israel
KARAKOÇ, Ömer Cem, Çankırı
YURTCAN, Murat, Edirne
KARUT, Kamil, Adana
ZCHORI-FEIN, E., Israel
KAŞKAVALCI, Galip, İzmir
ZEYBEKOĞLU, Ünal, Samsun
KAYDAN, Bora, Van
ZHANG, Y., Z.,, China
KAZAK, Cengiz, Adana
ZHAO, S., China
KAZAN, Kemal, Australia
İçindekiler (Contents)

Orijinal araştırmalar (Original articles)

Mortality effects of eicosanoid biosynthesis inhibitors on Spodoptera littoralis larvae co-injected
with the bacteria, Serratia marcescens
Eikosanoid biyosentezi inhibitörleri Serratia marcescens bakterisi ile birlikte Spodoptera littoralis
larvalarına uygulandığında larvalar üzerindeki ölüm etksi
Hasan TUNAZ, Mustafa KÜSEK ...................................................................................................................... 121-127

Determining phosphine resistance in rust red flour beetle, Tribolium castaneum (Herbst.)
(Coleoptera : Tenebrionidae) populations from Turkey
Türkiye Un Biti Tribolium castaneum (Herbst.) popülasyonlarında fosfin direncinin belirlenmesi
Erhan KOÇAK, David SCHLIPALIUS, Ramandeep KAUR, Andrew TUCK, Paul EBERT, Pat COLLINS,
Abdullah YILMAZ ............................................................................................................................................. 129-136

Insecticide resistance in two populations of Tuta absoluta (Meyrick, 1917) (Lepidoptera:
Gelechiidae) from Turkey
Türkiye'deki iki Tuta absoluta (Meyrick, 1917) (Lepidoptera: Gelechiidae) populasyonunda insektisit direnci
Melis YALÇIN, Serhan MERMER, Leyla Didem KOZACI, Cafer TURGUT ...................................................... 137-145

Comparation of Alticini (Coleoptera: Chrysomelidae: Galerucinae) species diversity in different
habitats selected from Bafa Lake Natural Park (Aydın) basin with a new record for Turkish fauna
Türkiye faunası için yeni bir kayıtla birlikte Bafa Gölü Tabiat Parkı (Aydın)’ndan seçilmiş farklı habitatların
Alticini (Coleoptera: Chrysomelidae: Galerucinae) tür çeşitliliğinin kıyaslanması
Fatma BAYRAM, Ebru Gül ASLAN .................................................................................................................. 147-157

Contributions to leafminer (Diptera: Agromyzidae) fauna and new records of plant pests and
weeds in Turkey
Türkiye galerisineği faunasına katkılar ve bitki zararlıları ve yabancı ot zararlılarına yeni kayıtlar
Oktay DURSUN, Hasan Sungur CIVELEK, Miroslav BARTÁK, Štěpán KUBÍK, Eyyüp Mennan YILDIRIM,
Miloš ČERNÝ ................................................................................................................................................... 159-169

Improvement of leaf miner [Liriomyza cicerina Rond. (Diptera: Agromyzidae)] resistance in Cicer species
Cicer türlerinde yaprak galeri sineğine [Liriomyza cicerina Rond. (Diptera: Agromyzidae)] dayanıklılığın geliştirilmesi
Cengiz İKTEN, Fatma Öncü CEYLAN, Cengiz TOKER ................................................................................... 171-178

Türkiye için yeni bir zararlı Ricania simulans (Walker, 1851) (Hemiptera: Ricaniidae)
Ricania simulans (Walker, 1851) (Hemiptera: Ricaniidae) a new pest for Turkey
Kibar AK, Şaban GÜÇLÜ, Cafer EKEN, Reyhan SEKBAN................................................................................... 179-186

Bademde yüksek konsantrasyonlarda ozon gazı uygulamasının Plodia interpunctella (Hübner)
ve Ephestia cautella (Walker)’ya karşı etkinliği
Efficacy of gaseous ozone at high concentrations against Plodia interpunctella (Hübner) and
Ephestia cautella (Walker) in Almond
Ali A. IŞIKBER, M. Serdar ÖZTEKİN, K. Sinan DAYISOYLU, Ahmet D. DUMAN, Selda EROĞLU ................ 187-198

Bozyazı ilçesi (Mersin) muz seralarında önemli bitki paraziti nematodların (Helicotylenchus
multicinctus, H. dihystera ve Meloidogyne spp.) (Nemata) popülasyon değişimlerinin araştırılması
Investigation on population dynamics of important plant parasitic nematodes (Helicotylenchus multicinctus,
H. dihystera and Meloidogyne spp. (Nemata) in banana greenhouses grown in Bozyazı (Mersin)
Ece B. KASAPOĞLU, Gürkan YORAZ, İbrahim Halil ELEKÇİOĞLU ............................................................... 199-207

Biber hat ve çeşitlerinin Meloidogyne incognita’ya karşı dayanıklılığı
Resistance of pepper lines against Meloidogyne incognita
Adem ÖZARSLANDAN, Hasan PINAR, Atilla ATA, Davut KELEŞ .................................................................. 209-215
Türk. entomol. derg., 2015, 39 (2): 121-127 ISSN 1010-6960
DOI: http://dx.doi.org/10.16970/ted.10222

Orijinal araştırma (Original article)
Mortality effects of eicosanoid biosynthesis inhibitors on
Spodoptera littoralis larvae co-injected with the bacteria, Serratia
marcescens1
Eikosanoid biyosentezi inhibitörleri Serratia marcescens bakterisi ile birlikte Spodoptera
littoralis larvalarına uygulandığında larvalar üzerindeki ölüm etksi
Hasan TUNAZ2* Mustafa KÜSEK2
Summary
The first step of the cellular defense reactions to bacterial, fungal and some viral infections in insects is
nodulation. We posed the hypothesis that Spodoptera littoralis, expresses melanoic nodulation reactions to bacterial
challenge and that injecting S. littoralis larvae with eicosanoid biosnthesis inhibitors (EBIs) plus bacteria would
increase larval mortality. Injecting larvae with EBIs, immediately before intrahemocoelic injections of the bacterium,
Serratia marcescens, sharply reduced the nodulation response to bacterial challenges. Separate treatments with
specific inhibitors including dexamethasone (a phospholipase A inhibitor), indomethacin, naproxen, ibuprofen,
2
(cyclooxygenase inhibitors), esculetin (a lipoxygenase inhibitor) and Phenidone (dual cyclooxgenase/lipoxygenase
inhibitor) impaired the ability of S. littoralis to form nodules in reaction to bacterial challenge. All concentrations of S.
marcescens alone, applied to S. littoralis, caused low mortality of the larvae. However, an increased mortality of the
larvae was seen when S. marcescens was co-injected with the EBIs with different modes of action. These findings
support our hypothesis that virulent effects of entomopathogenic bacteria can be increased when S. littoralis immune
systems were suppressed.
Keywords: Insect cellular immunity, nodulation, eicosanoid, bacteria, Spodoptera littoralis
Özet
Böceklerde bakeri, fungus ve bazı virüs hastalıklarına karşı oluşan hücresel bağışıklıklardan ilki nodülasyon
reaksiyonudur. Bu çalışmada Spodoptera littoralis larvalarında Serratia marcescens bakterisine karşı oluşan
nodülasyon reaksiyonu, bakterinin larvalar üzerindeki ölüm etkisi ve bakteri ile birlikte eiokosanoid biyosentezi
inhibitörleri larvalara uygulandığında larvaların ölüm oranını etkileyip etkilemeyeceği test edilmiştir. S. littoralis
larvalarına bakteri uygulamasında hemen önce eiokosanoid biyosentezi inhibitörleri enjekte edildiğinde böceklerede
bakteriye karşı oluşan nodül sayısında önemli derecede azalma olmuştur. Tüm S. Marcescens bakteri
konsantrasyonları larvalara tek başına uygulandığında düşük oranda larva ölümü ortaya çıkmıştır. Diğer taraftan S.
marcescens bakterisi ile birlikte eikosanoid biyosentezi inhibitörleri S. littoralis larvalarına uygulandığında larvalar
üzerindeki ölüm oranı yalnız bakteri uygulanan larvalara oranla önemli derecede yükselmiştir. Bu bulgular böceklerin
bağışıklık sistemi baskı altına alındığında, S. marcescens bakterisinin entomopatojen etkisinin arttığını göstermiştir.
Anahtar sözcükler: Böcek hücresel bağışıklığı, nodülasyon, eikosanoid, bakteri, Spodoptera littoralis

1 This study was supported by The Scientific and Technological Research Council of Turkey (TUBİTAK Project no: 110O159)
2 KSÜ, Faculty of Agriculture, Plant Protection Department, Kahramanmaraş/TURKEY
* Sorumlu yazar (Corresponding author) e-mail: [email protected]
Alınış (Received):12.12.2014 Kabul ediliş (Accepted): 17.03.2015 Çevrimiçi Yayın Tarihi (Published Online): 06.04.2015
121
Mortality effects of eicosanoid biosynthesis ınhibitors on Spodoptera littoralis larvae co-injected with the bacteria, Serratia marcescens
Introduction
Insect defense systems to microbial infection produce humoral and hemocytic (cellular) reactions
(Gillespie et al., 1997; Stanley, 2000; Satyavathi et al., 2014; Stanley & Kim, 2014). Humoral reactions,
antibacterial proteins, such as cecropins, attacins, diptericins, and defensins, take hours for their full
expression (Leulier et al., 2003; Stanley & Miller, 2006). On the other hand, hemocytic responses are very
quick, typically occur within minutes of an infection cycle and involve direct interactions between
circulating hemocytes and infecting microbes (Stanley & Miller, 2006; Satyavathi et al., 2014). Specific
cellular defense mechanisms include phagocytosis, nodulation and encapsulation (Strand, 2008).
Eicosanoids are oxygenated metabolites of arachidonic acid and two other polyunsaturated fatty
acids, the structures and biosynthesis of which are outlined elsewhere (Stanley, 2000; Stanley & Kim,
2014). Eicosanoids are very well understood in the contexts of human and animal medicine, where they
mediate many pathophysiological events, such as inflammation. Eicosanoids are also important for many
actions in invertebrates, as reviewed (Stanley, 2000; Stanley & Kim, 2014).
Stanley-Samuelson et al. (1991) first time showed that eicosanoids mediate one or more cellular
reactions responsible for clearing bacterial infections from hemolymph circulation. After this work, more
detailed research was done to determine which of the several cellular defense reactions depend on
eicosanoid biosynthesis. Miller et al. (1994) hypothesized that eicosanoids mediate nodulation reactions
to bacterial infections. After these findings, Stanley and his colleagues developed the hypothesis that
eicosanoids mediate nodulation reactions to bacterial infections in most, if not all, insect species, now
known as the eicosanoid hypothesis (Stanley, 2000). Several research groups have tested the hypothesis
for many insect species, as summarized in reviews (Stanley, 2006; Stanley & Miller, 2006; Stanley & Kim,
2014). All experimental work has strongly supported the idea.
Howard et al. (1998) tested the influence of bacterial species on nodule formation in insects. Their
result showed that nodulation intensity varies according to the species of infecting bactaria. Mandato et
al. (1997) found that cell spreading, a distinct phase of nodulation, and phagocytosis are mediated by
eicosanoids in wax moths, Galleria mellonella. The eicosanoid hypothesis is also supported by another
line of work on humoral immunity. Morishima et al. (1997) found that biosynthesis of anti-bacterial
proteins also depends on eicosanoids in the silkworm, Bombyx mori. The other role of eicosanoids in
insect cellular immunity were tested by Dean et al. (2002) and Lord et al. (2002). They suggested that
besides bacteria, eicosanoids mediate Manduca sexta cellular response to the fungal pathogens,
Beauveria bassiana and Metarhizium anisopliae. These findings uniformly support the eicosanoid
hypothesis. Connick et al. (2001) tested the role of eicosanoid biosynthesis inhibitors when co-applied
with pathojen bacteria, Serratia marcescens for insect pest control. Their results showed that increased
mortality of the termites, Coptotermes formosanus was seen when the bacteria were co-applied with
eicosanoid biosytnhesis inhibitors. Similarly, Tunaz (2006) tested influence of different fungal species on
nodule formation and on mortality of Pieris brassicae larvae and to determine whether injecting P.
brassicae larvae with EBIs plus fungus would influence larval mortality. Again his result showed that
increased and faster mortality of P. brassicae larvae was seen when the fungi were co-applied with
eicosanoid biosytnhesis inhibitors. Moreover, Tunaz & Küsek (2012) showed that increased mortality of
Blattella germanica adults was seen when the bacteria, S. marcescens, were co-applied with eicosanoid
biosytnhesis inhibitors.
Therefore, the objectives of this study were to determine influence of the bacterium, S.
marcescens, on nodule formation and on mortality of larvae of S. littoralis and if injection of larvae with
EBIs plus the bacterium would kill the larvae faster or higher numbers than would the bacterium alone.
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Tunaz & Küsek, Türk. entomol. derg., 2015, 39 (2)
Materials and Methods
Organisms
Spodoptera littoralis were reared on a culture (38 g agar, 2600 ml distil water, 300 g corn flour, 120
g wheat embryo, 100 g yeast, 20 g casein, 14 g wesson salt, 8 g sorbic acid, 4 g nipagin, 600 mg
streptomisin, 18 g ascorbic asid and 80 mg vitamin complex) and maintained in the laboratory at 25±2 °C
and 65±5% relatıve humidity (RH). The larvae (5. instars) were tested for each bioassays at 25  2 oC
and 65 5 % RH.
The pathogen used is a non-pigmented strain of an entomopathogenic bacterium, Serratia
marcescens (Miller & Stanley, 1998). The bacteria were grown in 50 ml of nutrient agar in environmental
horizontal shaker at 37oC and 100 rev/min. The bacteria were grown at concentration of 109 cell/ml and
used 5 µl to the each insect from different concentration of 109, 108, 107,106 cell/ml.
Reagents
The phospholipase A (PLA ) inhibitor dexamethasone {(11, 16)-9- fluoro-11,17,21-trihydroxy-
2 2
16-methylpregna-1,4-dione}, the cyclooxygenase inhibitors ibuprofen {a-methyl-4(2-methylpropyl)
benzeneacetic acid}, indomethacin {1-P-(chlorobenzyl)-5-methoxy-2-methyl-3-indolyl-acetic acid} and
naproxen {O-2-(6-methoxy-naphthyl) propionic acid}, the dual cyclooxygenase and lipoxygenase inhibitor
phenidone {1-pheny-3-pryazolidinone}, and the 5- and 12- lipoxygenase inhibitor, esculetin {6,7-
dihydroxycoumarin} were all purchased from Sigma Chemical Co. (St. Louis, MO).
Influence of eicosanoid biosynthesis inhibitors on nodulation
We divided larvae of S. littoralis into groups and injected individuals in each group with either the
phospholipase A inhibitor dexamethasone, the cyclooxygenase inhibitors indomethacin, naproxen,
2
ibuprofen, and the dual cyclooxygenase and lipoxygenase inhibitor phenidone, or lipoxygenase inhibitor
esculetin, all in standard dosages of 104 µg in 4 µl of ethanol. Control insects were injected with 4 µl of
ethanol. Following injections, the larvae of the S. littoralis were infected with 109 S. marcescens cell in 5 µl
saline using a 50 µl Hamilton 701 micro-syringe. Control insects were injected with 5 µl saline containing
(0.85 % NaCl). All the injections were applied into one side of abdomen (laterally just under the cuticle) of
S. littoralis larvae for injecting directly into the hemolymph circulation. Each test was replicated three
times and ten larvae were used for each replicate. At 6 hour post injection (hpi), the larvae of the S.
littoralis were anesthetized and nodulation was assessed. For nodulation assessment, larvae of the S.
littoralis were anesthetized by chilling on ice, then their hemocoels were exposed. Melanized, brownish
black nodules were counted under a stereo microscope at 45x. The nodules were distinct, and direct
counting reliably reflected the extent of the nodulation response to infections. After the first counting, the
alimentary canal was removed. Nodules in the previously unexposed areas and remaining internal tissues
were then counted.
Influence of the bacteria concentrations on mortality of S. littoralis larvae
Different concentrations (109, 108, 107,106 cell/ml) of the bacteria were injected into one side of
abdomen (laterally just under the cuticle) of S. littoralis larvae using a 50 µl Hamilton 701 micro-syringe.
The larvae of S. littoralis (10 larvae for each concentration of bacterial cell) were injected with different
concentration of S. marcescens cell in 5 µl saline using a 50 µl Hamilton 701 micro-syringe. Control
insects were injected with 5 µl saline containing (0.85 % NaCl). Each test was replicated three times and
ten larvae were used for each replicate. After injection the larvae were kept on room temperature.
Mortality was assessed during the seven days after injections. Mortality was identified as the larvae
unable to move when placed on their dorsal side and unable to respond to prodding.
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Mortality effects of eicosanoid biosynthesis ınhibitors on Spodoptera littoralis larvae co-injected with the bacteria, Serratia marcescens
Effects of eicosanoid biosynthesis inhibitors on mortality of S. littoralis larvae when co-
injected with the bacteria
Spodoptera littoralis larvae were divided into groups and individuals in each group were injected
with either PLA inhibitor dexamethasone, three of the cyclooxygenase inhibitor, naproxen, indomethacin
2
and ibuprofen, the dual cyclooxygenase and lipoxygenase inhibitor phenidone, or the lipoxygenase
inhibitor esculetin, all in standard dosages of 104 µg in 4 µl EtOH. Control insects were injected with 4 µl
EtOH. Following injections, the larvae were injected with 109 S. marcescens cell. Each test was replicated
three times and ten larvae were used for each replicate. After injection the larvae were kept on room
temperature as described. Mortality was assessed at selected times after injections as described above.
Statistical Analysis
Data on nodulation and mortality were analyzed using the General Linear Models procedure, and
mean comparisons were made using Least Significant Difference (LSD) test (p  0.0001) (SAS Institute
Inc., 1989).
Results
Influence of eicosanoid biosynthesis inhibitors on nodulation
Figure 1 shows that, compared to control (EtOH) larvae of S. littoralis, the nodulation response to
bacterial infections was significantly reduced in all experimental S. littoralis groups (LSD, p<0.0001).
There were no significant differences among the effects of individual inhibitors.
160
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u 60
N b
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20
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Eth+B Esc+B Nap+B Phe+B Dex+B Ind+B Ibu+B
Treatments

Figure 1. Effect of treating Spodoptera littoralis larvae with individual eicosanoid biosynthesis inhibitors on nodule formation in response to
infections with, Serratia marcescens. Each point indicates the mean number of nodules found in each insect, and the error bars
represent ± SEM. Histogram bars with same letter are not significantly different from each other (LSD, p  0.01).
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Tunaz & Küsek, Türk. entomol. derg., 2015, 39 (2)
Concentration-response for S. marcescens on mortality of S. littoralis larvae
Table 1 indicates that the mortality due to S. marcescens cell was relatively low with all the
concentrations of the bacteria. Although experimental data do not show in a strictly linear manner, we
recorded increased mortality with increased concentrations of S. marcescens and increased time, from
approximately 7 % mortality on day 1 to 20 % mortality on day 7 at 106 cell/larvae and at 108 cell/larvae,
larval mortality reached a high of 30 % by day 7. At the highest concentrations, 108 and 109 cell/larvae, we
recorded 23-30 % mortality after 7 days, which is significanly diffrent (LSD, p<0.0001) than mortality of
the control larvae (3 % mortality by day 7).
Table 1. The influence of increasing concentration of Serratia marcescens cell on % mortality of Spodoptera littoralis with respect to
time (days)(± standart error)
Mortality (%) in Time
Bacterial concentration

1st day 2nd day 3rd day 4th day 5th day 6th day 7th day

109 cell/ml 0±0a 6.6±6.6ab 6.6±6.6ab 10±5.7ab 10±5.7ab 13.3±3.3b 23.3±13.3b

108 cell/ml 6.6±6.6a 13.3±6.6b 13.3±6.6b 16.6±8.8b 16.6±8.8b 20±5.7b 30±11.5b

107 cell/ml 0±0a 3.3±3.3a 3.3±3.3a 3.3±3.3a 3.3±3.3a 6.6±3.3a 6.6±3.3a

106 cell/ml 6.6±3.3a 10±0b 10±0b 13.3±3.3b 16.6±6.6b 20±5.7b 20±5.7b

Control (saline) 3.3±3.3a 3.3±3.3a 3.3±3.3a 3.3±3.3a 3.3±3.3a 3.3±3.3a 3.3±3.3a
* Means in the same column followed by the same letters are not significantly different (P<0.0001) as determined by LSD-test.
Effects of co-injected bacterial cell and EBIs on mortality of S. littoralis larvae
EBIs strongly enhanced absolute mortality and the speed of kill due to bacterial challenge (Table
2). High mortality (66-93 %) obtained in larvae treated with the all inhibitors plus the concentration (109
cell/larvae) of bacteria by 24 hpi. Very low mortality (0-13 %) was recorded in controls (only bacteria and
EtOH+ bacteria) at 24 hpi. Co-injections of some of EBIs plus bacteria led to quite high mortality of
larvae, from 87 % mortality with ibuprofen to over 90 % mortality with dexamethasone and esculetin by 24
hpi, whereas the control larvae produced significantly less mortality at 24 hpi (LSD, p<0.0001).

Table 2. Effect of eicosanoid biosynthesis inhibitors on% mortality of Spodoptera littoralis larvae infected with Serratia marcescens(±
standart error)
Eicosanoid biosynthesis inhibitors+bacterial Mortality (%) in Time
concentration
6 hours 24 hours
Only bacteria (109 cell/ml) 0±0a 0±0a
EtOH+ bacteria (109 cell/ml) 0±0a 13.3±6.6b
Phenidone+ bacteria (109 cell/ml) 6.6±6.6a 66.6±17.6c
Naproxen+ bacteria (109 cell/ml) 6.6±6.6a 80±11.5cd
Esculetin+ bacteria (109 cell/ml) 6.6±6.6a 93.3±6.6d
Dexamethasone+ bacteria (109 cell/ml) 6.6±6.6a 93.3±6.6d
Indomethacin+bacteria (109 cell/ml) 0±0a 73.3±6.6c
Ibuprofen+ bacteria (109 cell/ml) 6.6±6.6a 86.6±13.3cd
* Means in the same column followed by the same letters are not significantly different (P<0.0001) as determined by LSD-test.
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Mortality effects of eicosanoid biosynthesis ınhibitors on Spodoptera littoralis larvae co-injected with the bacteria, Serratia marcescens
Discussion
The data reported in this paper strongly support the idea that EBIs significantly increased insect
larval mortality due to pathogenic bacterial challenge. First, six different eicosanoid biosynthesis
inhibitors significantly reduced nodulation when compared to control treatments. Second, relative to
control larvae, the bacteria caused relatively low larval mortality. Moreover, treating larvae with any of six
EBIs substantially increased mortality due to bacterial challenge. We infer from these data that disabling
immune signaling by inhibiting eicosanoid synthesis renders insects unable to protect themselves from
bacterial challenge and that the lack of immune protection is lethal.
The idea that eicosanoids mediate insect cellular immunity was first suggested by Stanley-
Samuelson et al. (1991). They showed that when eicosanoid biosynthesis is inhibited by EBIs, M. sexta
larvae could not clear the pathogenic bacterium, S. marcescens from their hemolymph. More important,
this situation increased insect mortality. Since after this first paper, several laboratory groups have
indicated eicosanoids are involved in nodulation formation (Stanley, 2006; Stanley & Miller, 2006; Stanley
& Kim, 2014). There is now considerable evidence for the involvement of eicosanoids in insect immune
reactions to bacteria, fungal, protozoan and parasitoid challenge in a wide range of insects. Dean et al.
(2002) and Lord et al. (2002) tested the hypothesis that eicosanoids mediate nodulation reactions to
fungal infection in M. sexta. They found that eicosanoids act in M. sexta nodulation response to B.
bassiana and M. anisopliae. Connick et al. (2001) suggested that there were synergestic effects of EBIs
with the bacterium, S. marcescens on mortality of termites; Coptotermes formosanus. Similarly, Tunaz
(2006) tested influence of different fungal species on nodule formation and on mortality of P. brassicae
larvae and to determine whether injecting P. brassicae larvae with EBIs plus fungus would influence larval
mortality. Again his result showed that increased and faster mortality of P. brassicae larvae was seen
when the fungi were co-applied with eicosanoid biosytnhesis inhibitors. Additionally, Tunaz & Küsek
(2012) showed that increased mortality of B. germanica adults was seen when the bacteria, S.
marcescens were co-applied with eicosanoid biosytnhesis inhibitors. Similar to these works, we
suggested that there were synergestic effects of EBIs with the bacterium, S. marcescens on mortality of
S. littoralis. The bacterial concentration-response experiment (S. marcescens) on larval mortality showed
that mortality was relatively low without EBIs.
The pharmacological chemicals we used inhibit different eicosanoid biosynthetic pathways in
mammals. Dexamethasone inhibits phospholipase A which releases arachidonic from membrane
2
phospholipids; naproxen, indomethacin and ibuporfen inhibit cyclooxygenase; esculetin inhibits
lipoxygenase; and phenidone inhibits both cyclooxygenase and lipoxygenase (Stanley, 2000). Hence,
because all the inhibitors we tested enhanced the susceptibility of S. littoralis to the injected bacteria, it is
possible that both cyclooxygenase and lipoxygenase pathways are involved in mediating the
immunomodulatory effects of eicosanoids in this insect. Finally, the results supported the hypothesis.
Eicosanoid biosynthesis inhibitors led to increased larval mortality, which supports the concept that
biological control programs can be enhanced by engineering gene-silencing constructs into crop plants.
Acknowledgement
The authors thank the Scientific and Research Council of Turkey (Ankara) for financial support.
126
Tunaz & Küsek, Türk. entomol. derg., 2015, 39 (2)
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